DEVELOPMENT AND EVALUATION OF Pleurotus

pulmonarius MYCELIUM AS ENCAPSULATED LIQUID

SPAWN FOR CULTIVATION

NORJULIZA BINTI MOHD KHIR JOHARI

FACULTY OF SCIENCE

UNIVERSITY OF MALAYA

KUALA LUMPUR

2019

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DEVELOPMENT AND EVALUATION OF Pleurotus pulmonarius MYCELIUM AS ENCAPSULATED LIQUID

SPAWN FOR CULTIVATION

NORJULIZA BINTI MOHD KHIR JOHARI

DISSERTATION SUBMITTED IN FULFILMENT

OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

INSTITUE OF BIOLOGICAL SCIENCES FACULTY OF SCIENCE

UNIVERSITY OF MALAYA KUALA LUMPUR

2019

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DEVELOPMENT AND EVALUATION OF Pleurotus pulmonarius

MYCELIUM AS ENCAPSULATED LIQUID SPAWN FOR CULTIVATION

ABSTRACT

Pleurotus pulmonarias also known as grey oyster mushroom is the most popular edible

mushroom in Malaysia. Typically, commercial cultivators use grain spawn to inoculate

into steriled fruiting substrate but contamination rate and attraction to pest during spawn

running is high. Good quality spawn should be free from diseases, fast colonisation with

high yield potential. Therefore, to overcome the low quality spawn problems, the

application of submerged culture technology followed by encapsulation of mycelium is

proposed. Growers can easily utilize the spawn using this technology without the need

for an expensive spawn inoculator equipment. The aim of this study is to optimize

production and delivery of P. pulmonarius mycelium to be used as spawn for cultivation.

An optimised culture medium at initial pH of 5.5 consisting of brown sugar, 2%; baker

yeast, 1%; spent grain extract, 1%; potassium dihydrogen phosphate (KH2PO4), 0.05%;

dipotassium hydrogen phosphate (K2HPO4), 0.05%; magnesium sulfate (MgSO4.7H20),

0.05% and Tween 80, 0.5% successfully supported high growth of 11.89 ±3.80 g/ L dried

mycelial biomass rapidly in 60 hours at 28°C in a 2-L stirred tank bioreactor. The

optimum storage time determined for soluble starch P. pulmonarius encapsulated

mycelial broth (SS-PPEMB) was 10 days stored in sterile distilled water at 4°C (C4

condition) exhibiting 100% germination on sawdust fruiting substrate. The potential of

SS-PPEMB as spawn was assessed on sawdust fruiting substrate in polyethylene bags

and the spawn run rate observed was 3.80 mm/day with biological efficiency of 180.99 ±

13.16 % even after storage at 10 days. As a conclusion, this study has successfully

produced a high quality spawn with low risk of contamination and high yield. This

technology is also applicable to other species of mushroom cultivated by mushroom

industry.

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Keyword: Mushroom liquid inoculum, liquid fermentation, encapsulated mycelium, mushroom cultivation

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PERKEMBANGAN DAN PENILAIAN MISELIUM Pleurotus pulmonarius

SEBAGAI BENIH CECAIR TERENKAPSULAT DALAM PENANAMAN

CENDAWAN

ABSTRAK

Pleurotus pulmonarius juga dikenali sebagai cendawan tiram kelabu merupakan

cendawan yang boleh dimakan paling digemari di Malaysia. Pengusaha cendawan

lazimnya menggunakan benih gandum untuk disuntik ke bongkah yang steril tetapi

menghadapi risiko pencemaran yang tinggi di samping menarik haiwan perosak semasa

pengeraman. Ciri-ciri benih yang berkualiti tinggi ialah bebas dari pencemaran dan

pertumbuhan yang cepat serta berpotensi mengeluarkan hasil tuaian yang tinggi. Oleh

yang demikian untuk mengatasi masalah benih yang tidak berkualiti, aplikasi teknologi

kultur tenggelam diikuti dengan enkapsulasi miselium dicadangkan. Pengusaha dapat

menggunakan benih dengan mudah melalui teknologi ini tanpa memerlukan penyutikan

benih yang mahal. Matlamat kajian ini adalah untuk mengoptimum penghasilan miselium

dan pengedaran miselium untuk digunakan sebagai benih dalam penanaman cendawan.

Kultur medium dengan pH awal 5.5 yang telah dioptimumkan terdiri dari gula perang,

2%; yis roti, 1%; ekstrak bijirin buangan, 1%; kalium dihidrogen fosfat (KH2PO4), 0.05%;

dikalium hidrogen fosfat (K2HPO4), 0.05%; magnesium sulfat (MgSO4.7H20), 0.05% dan

Tween 80 berjaya menampung pertumbuhan yang tinggi 11.89 ± 3.80 g/L biomasa

miselium kering dalam masa yang cepat 60 jam pada 28°C dalam tangki bioreaktor 2-L

yang dikacau. Masa penyimpanan optimum yang ditentukan untuk kaldu miselia P.

pulmarius terenkapsulat dengan kanji terlarut (SS-PPEMB) adalah 10 hari yang disimpan

dalam air suling yang steril pada suhu 4°C (keadaan C4) dengan 100% percambahan atas

medium habuk kayu. Potensi SS-PPEMB sebagai benih dinilai menggunakan medium

habuk kayu dalam bag polietilena menunjukkan kadar pertumbuhan miselium sebanyak

3.80 mm/hari dan kadar kecekapan biologi 180.99 ± 13.16%. walaupun selepas

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penyimpanan 10 hari. Kesimpulannya, hasil kajian ini berjaya menghasilkan benih

cendawan yang berkualiti tinggi dengan risiko pencemaran yang rendah dan hasil yang

tinggi. Teknologi ini dapat diaplikasikan kepada penanaman spesies cendawan yang lain

dalam industri cendawan.

Katakunci: Cendawan, cecair inokulum, penapaian cecair, miselium terenkapsulat, penanaman cendawan

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ACKNOWLEDGEMENTS

All praise is due to Allah, the Almighty, and the Lord of all worlds who gave me

the strength and courage to complete my study. I wish to express my heartfelt gratitude

to my family for their unconditional love, endless support and encouragement.

My utmost gratitude to my supervisor, Prof. Dr. Noorlidah Abdullah, who has

steadfast supported me throughout my study with her guidance, patience and knowledge

as well as her unselfish and unfailing support as I hurdle all the obstacles in the completion

of my study.

Special thanks to Erlina Abdullah, Noor Hasni Mohd Fadzil, Amal Rhaffor, Siti

Norfaizah Mohd Ismail, Rushitha Sadasevam, Nur Edahyu and Amalina Amirullah for

your friendship and encouragements. I would like to take this opportunity to dedicate my

deepest appreciation to all my friends in the Mycology Lab for their support and guidance

and most importantly for contributing their unselfish time in sharing wonderful ideas and

helping me carry out my research works.

Thank you to each and everyone who have helped me during my journey to

complete my research and this thesis.

Norjuliza Mohd Khir Johari

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TABLE OF CONTENT

ABSTRACT .............................................................................................................. iii

ABSTRAK .................................................................................................................. v

ACKNOWLEDGMENTS ....................................................................................... vii

TABLE OF CONTENT ......................................................................................... viii

LIST OF FIGURES .................................................................................................. xi

LIST OF TABLES .................................................................................................. xiv

LIST OF SYMBOLS AND ABBEREVIATIONS ................................................ xv

LIST OF APPENDICES ....................................................................................... xvii

CHAPTER 1.0: INTRODUCTION ......................................................................... 1

CHAPTER 2.0: LITERATURE REVIEW ............................................................. 5

2.1 Overview of mushroom cultivation industry ................................................... 5

2.2 Stages of mushroom cultivation of Pleurotus species ..................................... 7

2.2.1 Fruiting substrate preparation ................................................................. 9

2.2.2 Spawning .............................................................................................. 10

2.2.2.1 Grain spawn ..................................................................................... 12

2.2.2.2 Liquid spawn .................................................................................... 15

2.2.2.3 Submerged fermentation of mycelium to produce liquid spawn and encapsulation medium ........................................................................... 17

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2.2.2.4 Spawn quality ................................................................................... 21

2.2.3 Spawn Run/ Incubation ........................................................................ 21

2.2.4 Fruiting phase ....................................................................................... 22

2.3 Pleurotus pulmonarius .................................................................................. 22

2.4 Cultivation of P. pulmonarius in Malaysia ................................................... 25

CHAPTER 3.0: MATERIALS AND METHODS ................................................ 28

3.1 Preparation of P. pulmonarius mycelial culture ............................................ 28

3.2 Optimization of growth medium formulation for mycelial growth of P. pulmonarius by submerged fermentation ...................................................... 29

3.2.1 Optimization of brown sugar concentration and yeasts concentration ............................................................................................................. 29

3.2.2 Effect of supplementation of spent grain extract, minerals and Tween 80 in the liquid medium ........................................................... 31

3.2.3 Effect of pH of optimised growth medium ......................................... 32

3.3 Production of P. pulmonarius mycelium in optimal medium using a 2l-automated stirred bioreactor .......................................................................... 32

3.4 Non-supplemented encapsulation of P. pulmonarius mycelial broth (MB) . 34

3.5 Optimization of P. pulmonarius encapsulated mycelium broth (PPEMB) ... 36

3.5.1 Optimization of encapsulation solution by supplemented with mashed potato and soluble starch at various concentrations .............. 36

3.5.2 Effect of supplementation of 5% mashed potato and 5% soluble in encapsulation solution on sporophore yield ........................................ 36

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3.5.3 Evaluation of viability of soluble starch (SS)-PPEMB after storage at various conditions ............................................................................... 37

CHAPTER 4.0: RESULTS & DISCUSSION ....................................................... 40

4.1 Optimization of growth medium formulation for the production of P. pulmonarius mycelium .................................................................................. 40

4.1.1 Effect of brown sugar concentration, nitrogen sources and concentration ....................................................................................... 40

4.1.2 Effect of supplementation of spent grain extract, minerals and Tween 80 .............................................................................................. 43

4.1.3 Effect of pH of growth medium ........................................................... 48

4.2 Scale-up production of P. pulmonarius mycelium using a 2-L automated bioreactor ....................................................................................................... 49

4.3 Effect of fermentation duration/ harvesting time mycelium in a bioreactor on germination and storage time of P. pulmonarius non-supplemented PPEMB .......................................................................................................... 51

4.4 Optimization of P. pulmonarius Encapsulation solution to prepare PPEMB ......................................................................................................... .53

4.4.1 Optimization of encapsulation solution by supplemented with mashed potato and soluble starch at various concentration [Nutrient supplemented (NS)-PPEMB] ............................................................. 53

4.4.2 Effect of supplementation of 5% mashed potato and 5% soluble starch in encapsulation solution on sporophore yield ......................... 56

4.4.3 Evaluation of viability of soluble starch (SS)-PPEMB after storage at various conditions ........................................................................... 57

CHAPTER 5.0: CONCLUSION & FUTURE RECOMMEDATIONS ............. 64

REFERENCES ........................................................................................................ 66

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APPENDICES ......................................................................................................... 84

Appendix A: Experimental ........................................................................................ 84

Appendix B: Raw Data .............................................................................................. 86

Appendix C: Publication ........................................................................................... 87

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LIST OF FIGURES

Figure Titles Page

Figure 1.1: Average production share of mushroom and truffles by region from 1994-2016

2

Figure 2.1: Process flow of mushroom cultivation 8

Figure 2.2: Polyethylene or polypropylene bags were covered with a plastic cover and a neck ring

10

Figure 2.3: Pleurotus pulmonarius spawn production process 14

Figure 2.4: Pleurotus pulmonarius a & b) A mature fruiting body was cultivated on sawdust substrate

24

Figure 3.1: Preparation of mycelial culture by tissue transfer 28

Figure 3.2: Two litre stirred tank bioreactor with the containing of 10% (v/v) of liquid inoculum in medium of 2% brown sugar, 1% baker’s yeast, 1% spent grain extract, 0.5% Tween 80, 0.5% MgSO4, 0.5% KH2HPO4 and 0.5% K2HPO4

34

Figure 3.3: The setup for for MB encapsulation. 35

Figure 4.1: Effect mycelial of P.pulmonarius supplemented a) with Tween 80 and b) without Tween 80 in shake-flask liquid fermentation medium

46

Figure 4.2: Mycelial biomass production (dry weight, g/L) of P. pulmonarius in 2-L automated bioreactor

50

Figure 4.3: Concentration of reducing sugar (mg/L) in the medium during growth of P. pulmonarius in an automated bioreactor

50

Figure 4.4: Growth rate of freshly prepared PPEMB on sawdust substrate in glass Petri dish at different harvesting time of 48 h and 60 h

52

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Figure 4.5: Germination of PPEMB on sawdust substrate after storage of 15 days.

52

Figure 4.6: Germination and colonisation of NS-PPEMB on sawdust substrate in glass Petri dish.

54

Figure 4.7: Growth rate of SS-PPEMB mycelium on sawdust substrate bag after different conditions of storage.

60

Figure 4.8: Biological efficiency of SS-PPEMB on sawdust substrate bag after different condition of storage

60

Figure 4.9: Mycelial run of P. pulmonarius using grain spawn on fruiting sawdust substrate

62

Figure 4.10: Mycelial run of SS-PPEMB that when stored at C1 condition for 10 days on fruiting sawdust substrate

62

Figure 4.11: Mycelial run of SS-PPEMB that when stored at C2 condition for 10 days on fruiting sawdust substrate

62

Figure 4.12: Mycelial run of SS-PPEMB that when stored at C3 condition for 10 days on fruiting sawdust substrate

63

Figure 4.13: Mycelial run of SS-PPEMB that when stored at C4 condition for 10 days on fruiting sawdust substrate

63

Figure 4.14: Sporophore yield of SS-PPEMB on fruiting sawdust substrate inoculated with SS-PPEMB stored at C1- C4 condition for 5 days and 10 days

63

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LIST OF TABLE Table Titles Page

Table 3.1: The percentage of carbon and nitrogen sources concentration in liquid fermentation medium

30

Table 3.2: The percentage concentration of supplementation of spent grain extract, and minerals in shake-flask liquid fermentation medium

32

Table 4.1: Effect of brown sugar concentration, nitrogen sources and concentration on mycelial dry weight of P. pulmonarius in shake-flask liquid fermentation medium

42

Table 4.2: Effect of spent grain extract, minerals and Tween 80 on the mycelial dry weight by P. pulmonarius in shake-flask liquid fermentation medium

47

Table 4.3: Effect of initial pH on the average mycelial dry weight by P. pulmonarius in shake-flask medium culture

48

Table 4.4: Percentage of germination of non- supplemented PPEMB on sawdust substrate in glass Petri dish.

51

Table 4.5: Percentage of germination NS-PPEMB on sawdust substrate in glass Petri dish plate.

55

Table 4.6: Growth rate (mm/day), total sporophore yield (g) and biological efficiency (%) of NS-PPEMB.

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Table 4.7: Growth rate (mm/day), total sporophore yield (g) and biological efficiency (%) of SS-PPEMB in tested storage condition and storage life

59

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LIST OF SYMBOLS AND ABBREVIATIONS

% : Percentage

°C : Degree Celsius

± : Plus-minus

BE : Biological efficiency

BKY : Baker Yeast

BS : Brown sugar

CaCl2 : Calcium chloride

CaCO3 : Calcium carbonate

cm : Centimetre

CO2 : Carbon dioxide

DNS : 3,5-dinitrosalicylic acid

EM : Encapsulated mycelium

EMB : Encapsulated mycelium broth

et al. : And other

g : Gram

g/L : Gram per litre

h : Hour

hrs : Hours

H2SO4 : Sulfuric acid

K2HPO4 : Dipotassium hydrogen phosphate

KH2PO4 : Potassium dihydrogen phosphate

L : Litre

M : Molar

MB : Mycelial broth

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mg : Milligram

MgSO4.7H2O : Magnesium sulfate heptahydrate

min : Minute

mL : Millilitre

mm : Millimetre

NaoH : Sodium hydroxide

NO : Number

NS-PPEMB : Nutrient supplemented Pleurotus pulmonarius encapsulated mycelium broth

PPEMB : Pleurotus pulmonarius encapsulated mycelium broth

RB : Rice bran

rpm : Rotation per minute

SD : Sawdust

SG : Spent grain

SGE : Spent grain extract

SS-PPEMB : Soluble starch Pleurotus pulmonarius encapsulated mycelium broth

STD : Standard deviation

sp : Species

v/v : Volume per volume

w/v : Weight per volume

w/w : Weight per weight

YE : Yeast Extract

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LIST OF APPENDICES

APPENDIX A: Experimental Page

1.0 Flow chart of preparation of Malt Extract Agar (MEA) media

84

2.0 Determination of reducing sugar (DNS Method) 84

3.0 Preparation of sterile substrate in glass Petri Dish 85

4.0 Preparation of mashed potato 85

5.0 Preparation of sterile fruiting substrate in polyethylene bags

85

APPENDIX B: Raw Data 86

APPENDIX C: Publication 87

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CHAPTER 1

INTRODUCTION

Chang and Miles (1992) defined mushroom as a macrofungus with a distinctive

fruiting body, which can be either epigeous or hypogeus, and large enough to be seen

with the naked eye and to be picked by hand. Edible and medicinal mushrooms are

becoming valuable horticultural crop worldwide. Only around 35 species amongst the

300 species of edible mushroom that exist can be cultivated and around 20 species are

cultivated on an industrial scale (Sánchez, 2004). The popular mushroom that have been

commercially cultivated worldwide are Agaricus bisporus (button mushroom) followed

by Lentinula edodes (shiitake), Pleurotus spp. (oyster mushroom), Auricularia spp.

(wood ear mushroom), Flamulina velutipes (winter mushroom) and Volvariella volvacea

(straw mushroom) (Chang, 1999a).

Mushroom production is observed as the second most essential profitable

microbial technology behind yeast (Pathak et al., 2009). From Figure 1.1, the global

mushroom production by Asian countries is leading producing more than 69.1% of world

mushroom markets and then followed by Europe (22%). from total world mushroom in

2016 (FAO, 2016). Currently, 40% of total world edible mushroom are exported from

China and by this reason, China is known as the world’s biggest producer of mushroom.

Nevertheless, in China 95% of the total China production is for domestic consumption

(Zhang et al., 2014). In China, the government strongly boost and financially supported

the cultivation mushroom industry due to the benefit of mushroom as a source of quality

food. (Zhang et al., 2014) because of their high-quality protein; excellent unsaturated

fatty acids and high vitamins content available (Marshall & Nair, 2009; Kumar et al.,

2014; Valverde et al., 2015). The protein content in numerous mushrooms are about 19-

40% (dry weight) offering twice as much protein as vegetables and four times that of

oranges and can be a potential choice to meat (Jonathan et al., 2012; Bashir et al., 2014;

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Amuneke et al., 2017). China major consumption is Shiitake with 22.5%, followed by

Grey oyster mushroom 18.9% and Wood ear mushroom 16.8% (Li & Hu, 2014).

Figure 1.1: Average production share of mushroom and truffles by region from 1994-2016 (Adapted from http://www.fao.org/faostat/en/#data/QC/visualize). (Reprinted permission by FAO publication)

Pleurotus species are mostly cultivated particularly in south East Asia, India,

Europe and Africa, (Mandeel et al., 2005). Reis et al. (2012) stated that Pleurotus spp.

contains high levels of proteins, fibers, carbohydrates, vitamins and minerals, whereas

offering low calorie, fat and sodium levels. In Malaysia, mushroom is included as one

of the seven profit crops that are cultivated comprehensively (Ministry of Agriculture

Malaysia, 2011) and grey oyster is the most dominantly cultivated and marketed in

Malaysia (Haimid et al., 2013). Mushroom production total value in Malaysia has shown

tremendous increased from RM49.1 million in 2007 to RM79.0 million in 2011. In 2014,

the total value of production had further increased to more than RM110 million due to

the rising number of growers, land area and productivity (Department of Agriculture

Malaysia, 2015).

Mushroom production in Malaysia that has started in 1961 still shows a slow

increase due to many problems faced by the growers. One of the problem is the lack of

quality spawn. Spawn is defined as the substrate in which mushroom mycelium has grown

Asia 69.1%

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and which will be used as a “seed” in propagation for mushroom production. The genetic

and cultural characteristic of the mushroom species are carried in the spawn (Chang &

Miles, 2004). The quality of spawn affects the production yield and quality of mushroom

commercially. Chang and Miles (2004), stated that if the spawn has not been prepared

from a genetically proper fruiting culture or if a stock has degenerated or if it is too old,

the yield of mushrooms will be less than optimal. The intent and purpose of

inoculum/spawn is to promote the mycelium to a state of vigor where it can be launched

into bulk substrates. The substrate (solid or liquid) is not only used as a vehicle for evenly

spreading the mycelium at the same time it also acts as nutritional supplement (Staments,

1993).

Traditionally for mushroom production, solid inoculum using wheat grain

colonized by the fungal mycelium has been used (Hesseltine, 1987; Abe et al., 1992;

Gunde-Cimermam, 1999). According to Confrotin et al. (2008) the preparation of grain

spawn took longer growth period and stand higher risk of contamination compared to

liquid spawn. Certain findings showed that liquid spawn is a potential replacement of the

conventional grain spawn to be used in the various mushrooms cultivation (Kirchhoff &

Lelley, 1991; Friel & McLoughlin, 2000; Silveira et al., 2008).

However, currently it faces problems in inoculation since a sterile injector is

required which is expensive. Under non-aseptic conditions it may be contaminated since

its residual nutrients is a source of contamination. Meanwhile, certain mushroom such as

A. bisporus and L. edodes could not colonize on several cultivation substrates by using

liquid spawn (Leatham & Griffin, 1984; Friel & McLoughlin, 1999). In addition, this

problem will affect the yield potential of mushroom.

The encapsulated microbial enriched the capability of microorganisms under

storage or in opposing environmental conditions (Ortiz et al., 2017). Throughout

encapsulation process, the microorganisms are trapped to a micro-environment, this

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micro-environment potential be practically developed including complete nutrients to

preference the preservation or growth of microorganism (Khosravi et al., 2014). Hence,

this study is carried out to produce spawn in the form of encapsulated mycelium of a

popular edible mushroom, Pleurotus pulmonarius and to evaluate the performance for the

production of sporophores.

OBJECTIVES OF STUDY

1) To optimize medium formulation for submerged fermentation of P. pulmonarius

mycelium

2) To optimize immobilization of P. pulmonarius mycelium to be used as liquid

spawn and to assess the viability and storage life.

3) To evaluate the efficacy of immobilized mycelia on sawdust fruiting substrate.

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CHAPTER 2.0

LITERATURE REVIEW

2.1 Overview of mushroom cultivation industry

Mushroom is treasured as a precious natural ingredient recognised for their

therapeutic value, and even often used in religious rituals (Stille,1994) in the early

evolution of the Greeks, Egyptian, Romans, Chinese, and Mexicans (Chang & Miles,

2004).

Traditionally, edible mushroom was found to grow and harvested wild in the

forest of hilly areas, since it was initially difficult to domesticate and cultivate in

temperate and subtropical regions of the world (Shah et al., 2004; Zhang et al., 2014).

According to Van (2009), historically mushrooms have been collected to be consumed as

food, as hallucinogens and remedy or for utilitarian aspect such tinder. Even in this era,

particularly in southern Asia and other developing countries, picked/harvested edible

mushroom from wild woodlands is still essential as a source of food and medicinal (Arora,

2008; Yang et al., 2008; Fanzo et al., 2012). Fasola et al. (2007) reported that mostly

people in Nigerian depend on mushroom collected from the wild rather than commercial

mushroom production. Normally mushroom hunters collect different sporophores of

Nigerian mushroom and put on sale in local markets (Fasola et al., 2007).

There are various techniques and species in mushroom cultivation history. Around

600-700 A.D., China was the first to use wood logs method to cultivate Auricularia

auricula and L. edodes (Chang & Miles, 2004; Van, 2009). Later around 1600 year,

France cultivated A. bisporus by using composted substrate method. Consequently, in

Asia countries, mushroom species like L. edodes and Pleurotus spp. are famous and

produced in large scale and these make inroads into Western markets (Chang & Miles,

2004).

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Through 1990-2016, China, Italy, USA, Netherlands and Poland were the major

producers of the world’s mushrooms. The worldwide total production of mushrooms in

2016 was 10.790 million tonnes, which was a tremendous increase of nearly 521% from

the 2.071 million tonnes of mushrooms produced in 1990 (FAOStat 1990-2016).

According to the National Research Centre for mushroom, NRCM (2009), the percentage

of types of mushrooms cultivated internationally based on types are as follows: button

(31%), shiitake (24%), oyster (14%), black ear (9%), paddy straw (8%) and milky/ others

(14%). In 2013, the highest mushrooms in demand for consumption in China were the

Shiitake (22.5%), followed by grey oyster mushroom (18.9%) and the wood ear

mushroom (16.8%) (Li & Hu, 2014).

The technology of artificial mushroom cultivation is an integration of non-

traditional crops in the current agricultural techniques (Shah et al., 2004). Through the

innovation of mushroom science technology, mushroom cultivation can be implemented

in different regions of various countries. Mushroom cultivation is preferred by growers

due to the short period of harvest, utilisation of small plot of space and its contribution in

sustainable agriculture and forestry (Shah et al., 2004; Zhang et al., 2014). Mostly small

farmers in China, India and other developing countries gain benefits from mushroom

cultivation and the processed mushroom products in terms of financial, social and health

improvement (Shah et al., 2004).

Mushroom cultivation involves microbiology, composting technology,

environmental engineering, and marketing and management (Chang & Miles, 2004).

Edible mushrooms cultivation is an important modern practice in biotechnological

process that has potency for lignocellulosic organic waste recycling in order to decrease

of environmental pollution and produces food of protein-rich food and superior nutritious

value (Silva et al., 2002; Beetz & Kustudia, 2004; Sánchez, 2010;).

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Currently, mushroom cultivation industry is rising and new methods are being

developed with supportable research to boost the different numbers of cultivated

mushroom species production and mushroom products (Chang, 1999b; Lomberh et al.,

2002).

2.2 Stages of mushroom cultivation for Pleurotus species

In mushroom cultivation there are five stages that involves i) fruiting substrate

preparation, ii) spawning/ inoculation, iii) spawn run / incubation, iv) fruiting phase, v)

harvesting (Figure 2.1). On the other hand, according to Martínez-Carrera et al. (2000)

and Wang (1999), mushroom cultivation involved three major stages: (1) inoculum

(spawn) production, (2) substrate preparation, and (3) mushroom growing.

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Figure 2.1: Process flow of mushroom cultivation.

Fruiting substrate

preparation and sterilization

Spawning Spawn run / IncubationFruiting phase Harvesting

Preparation of spawn

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2.2.1 Fruiting Substrate Preparation

In Malaysia, the bag cultivation method is the most practical technique that most

mushroom growers implement in the preparation of fruiting substrates. This method

results in higher biological efficiency and shorter production cycle (Mata & Savoie,

2005). Through the process of fruiting substrate preparation, the carbon source and

nitrogen source are supplemented with minerals such as lime, calcium nitrate (Thevasingh

et al., 2005), gypsum, calcium carbonate (CaCO3) and calcium superphophate (Fan et al.,

2005). In addition, water is added and evenly mixed to raise the moisture content of the

substrate to 70-85%.

The principal ingredient of a basic fruiting substrate is normally various

agricultural waste such as sawdust, cottonseed hulls, wheat straw, paddy straw, coffee

residue and oil palm, as carbon source. Meanwhile, addition of nitrogen source is needed

such as rice bran, wheat bran, corn bran, and CaCO3 are also included (Shu-Ting & Philip,

2004). Other sources of cellulose, hemicellouse, and lignin may also be used in various

combinations with or without supplementation of nitrogen source. The prominent wood

moisture of the dry weight is in the range of 50%-60%. In practice, wood with 90-100%

water is acceptable, but may retard mycelial growth, which can lead to the low oxygen

content in the cells (Vintila et al., 1963).

In addition, Dietzler (1997) stated that the type of substrates, quantity (formulation

of substrate) and supplementation may affect some substrate abilities such as water

holding capacity and intensity of aeration; characteristics that consequently have an effect

on mushroom yield. There may be difficulties for the mycelium to colonize in a fruiting

substrate, if the substrate condition or type is too tight or too loose. Chen (2005) reported

that if the substrate is too wet, the air flow in the substrate will be clogged. If the water

collects at the bottom of the bag it may due to the substrate being too wet.

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After mixing substrate is packed into polyethylene or polypropylene bags, they

are covered with a plastic cover and a neck ring (Figure 2.2). The sterilization process of

fruiting bag is needed to remove competitive microbes and exterminate any

contamination. Several methods and parameters are used for the sterilization, and the

selection of these methods is dependent on several factors such as the nature of the bags,

bag size and amount of the substrate (Kim, 2005).

Figure 2.2: Polyethylene or polypropylene bags were covered with a plastic cover and a neck ring.

2.2.2 Spawning

The usage of pure culture from the stock mycelium growing on an agar plate is

not appropriate to be used directly as mushroom spawn. Therefore, it should be relocated

for growth on a suitable culture medium that is easy to handle, distribute, inexpensive and

convenient to inoculate (Chang & Miles, 2004). According to Chang and Miles (2004),

spawn was defined as the substrate in which mushroom mycelium had grown and which

would be used as a “seed” in propagation for mushroom production. The genetic and

cultural characteristic of the mushroom species are carried in the spawn (Chang, 2001;

Plastic cover

Neck ring

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Chang & Miles, 2004). It is a medium impregnated with mushroom mycelium. According

to Stanley and Awi-Waddu (2010), production of spawn is the first stage in mushroom

production. According to Sánchez (2010), the function of grain coated with mycelium is

to speed the mycelium to migrate to the specific bulk of the fruiting substrate.

This process of inoculating the spawn into the logs or cooled sterile fruiting

substrate in polyethylene bags is called spawning. It is important to make sure that the

mushroom spawn used is fresh, robust and not degenerated (Shu-Ting & Philip, 2004).

According to Miles and Chang (1997), there are several techniques of spawning,

depending on the cultivation technique. According to the method of applying a spawn on

composted substrate, the spawn is shattered into small pieces by crumbling it with fingers

and then spreading the pieces over the bed of the substrate surface. At the same time, it is

vital to ensure that the spawn is in good contact with the substrate, which can be done by

pressing it down firmly. Another technique of spawning is by using the fruiting bag, in

which the spawn is injected 2-2.5 cm deep into the substrate. In spawning, the most vital

aspect we should pay attention to is the amount of the spawn used per unit surface area,

and the larger the amounts of the spawn used the more rapid the substrate will be full with

mycelium (Miles & Chang, 1997). However, it is disadvantageous to use a large amount

of spawn per unit area, as it will result in higher cost for the additional spawn.

The inoculated bag of substrate is then incubated at 20 to 25°C. This is the

mycelial growth phase, in which the mycelium develops immediately after inoculation

(spawning) until the substrate is completely permeated with mycelium. Spawn running

(mycelial running) is the phase during which mycelium develops out from the spawn and

permeates the substrate. During the phase of spawn running, the mycelium secretes

enzymes to degrade complex substances in the substrate of bag and the mycelium

assimilates and conglomerates the needed nutrients in sufficient amounts required for

fruiting (Shu-Ting & Philip, 2004; Zadrazil et al., 2004).

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2.2.2.1 Grain Spawn

There are two types of spawn used in mushroom cultivation, which are the solid

and liquid spawns. According to Wang et al. (2011), there are many types of substrates

to prepare solid spawn such as:

i) grains spawn eg rye, sorghum, wheat, millet, or crushed corn;

ii) wood block or sawdust spawn eg hardwood sawdust especially oak, alder, cottonwood,

poplar, ash, elm, birch;

iii) straw spawn eg paddy straw or wheat straw.

Normally, mushroom growers will use solid spawns, such as the cereal grain spawn as

the conventional method due to the simple equipment preparations and techniques, its

competence to endorse the fruiting substrate faster and the ease of planting in this

technique (Abe et al., 1992; Friel & McLoughlin, 1999; Gunde-Cimermam, 1999).

However, this method also needs a large space for a long incubation period (Bahl, 1988;

Wang et al., 2011). In Malaysia, commonly spawn is grown on grains such as wheat,

crushed corn or millet. In addition, whole grain is also used since each kernel can become

a mycelia capsule, a platform from which mycelia can leap into the surrounding expanse.

One of the benefit from using smaller kernels of grain is that the grains offer more points

of inoculation per pound of spawn (Stamets, 2000). On the other hand, the preparation of

spawn for P. ostreatus cultivation is on grains such as wheat (Nwanze et al., 2005a;

Elhami & Ansari, 2008), corn (Nwanze et al., 2005a; Elhami & Ansari, 2008), millet

(Nwanze et al., 2005a; Elhami & Ansari, 2008; Narh et al., 2011), sorghum (Narh et al.,

2011) and other work on mixture of grain and grain straw (Muthukrishnan et al., 2000;

Sainos et al., 2006; Pathmashini et al., 2008).

The findings of Nwanze et al. (2005b) on grain selection showed that corn spawn

stimulated highest yield of fruiting Lentinus squarrosulus as compared to wheat and

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millet spawn. Elhami and Ansari (2008) also showed that corn spawn can contribute on

highest mycelia growth of oyster mushroom species (Pleurotus florida,

Pleurotus citrinopileatus and Pleurotus ostreatus) as compared to wheat and millet

spawn. Moreover, use larger grain of corn and wheat may promote more nutrient for

mycelia growth (Mottaghi, 2004).

The general method of grain spawn preparation includes grain washed with tap

water to remove dust and then it is soaked overnight. Then, the grain is formulated with

rice bran and calcium carbonate was added to adjust the pH and act as mineral. The grain

is then distributed in the autoclavable polyethylene bag (Figure 2.3a) and sterilized by

autoclaving for 1 hour at 121°C (Figure 2.3b). After the grains are cooled to room

temperature, they are inoculated with the plug of mycelium (Figure 2.3c). After a period

of around 2-3 weeks the grain is ready to be used as a spawn for mushroom cultivation

(Figure 2.3d).

From the point of view of Stamets (2000), sawdust spawn is far better than grain

spawn for the inoculation of outdoor mushroom cultivation because when grain spawn is

introduced to an outdoor bed, insects, birds and slugs quickly seek out the nutritious

kernels for food. By using sawdust spawn, it has the improvement of having more

particles or inoculation points per pound than does the grain, with more points of

inoculation and the mycelium colonization is also quicker (Stamets, 2000). Another

technique involves the use of wood plug spawn. This is done by inoculating the

mushroom mycelium into wedge-shaped pieces or cylindrical pieces of wood. After the

mycelium has fully colonised into the pieces of wood, these wood plugs can be used as

mushroom spawns. (Chang & Miles, 2004).

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Figure 2.3 : Pleurotus pulmonarius spawn production process : a) grain distribution into autoclavable polyethylene bags; b) sterilization by autoclaving for 1 h at 121°C; c) inoculation of cooled grain P. pulmonarius mycelial plugs; d) colonization of the grain with mycelium.

Certain species of mushroom are suitable for cultivation using the straw spawn

technique, such as the V. volvacea. In preparation of straw spawn (paddy straw), firstly

the paddy straw is soaked in water for 2 to 4 h, and then the water is drained drained and

the straw is cut into pieces of sized 2.5 to 5 cm long. Next it is mixed with 1% calcium

carbonate and 1% to 2% rice bran and is placed into clean wide-mouthed quart bottles

(Chang & Miles, 2004).

The problem that mushroom growers will have faced when using solid spawn is

the high risk of contamination by mitosporic fungi such as Trichoderma spp., also known

as the green mold disease (Hatvani et al., 2007). Normally Trichoderma spp is present in

the early stage of mushroom cultivation, especially during the spawning stage, but it can

also contaminate during the harvesting stage, which can cause huge losses in the

mushroom yield (Jandaik & Guleria, 1999). According to Ortiz et al. (2017), the

contamination of spawn may occur because of incorrect handling during the spawn

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preparation phase, or by intrinsic grain microbial contamination. Laca et al. (2006) and

Sreenivasa et al. (2010) stated that the population of microbial shift among grains batches

and the mitosporic fungi existing in cereals constitute a potential problem for spawn

quality.

2.2.2.2 Liquid Spawn

Mycelia or liquid spawn is an alternative method for generating spawn by

submerged fermentation. The implementation of liquid culture technology to the mycelia

production of higher fungi afford the possibility of industrial scale application to this

group of organisms (Humfeld, 1948). There are many advantages of adapting the liquid

spawn compared to grain spawn, as it produces higher yield of mycelium in compact

space, more uniform distribution mycelia biomass in shorter period, could lower

laboratory cost, and make the inoculation procedure smooth with reduced possibilities of

contamination and promote early fruiting (Chang & Miles, 1993; Eyal, 1991; Rosado et

al., 2002).

Liquid spawn can be inoculated directly to fruiting substrate and another method

is by encapsulation of the mycelium. On other hand, liquid spawn also makes it possible

for the mycelium to be propagated on solid support, encapsulation of mycelium or it can

be directly inoculated in the fruiting substrate (Friel & McLoughlin; 2000). According to

Kirchhoff and Lelley (1991), the use of liquid spawn for the sawdust substrate cultivation

lead to a higher fruiting body yield than grain spawn. Abdullah et al. (2013) reported that

liquid spawn of P. pulmonarius has the proficiency to colonise sterile sawdust substrate

in shortened time suggesting that the mycelium was spread more efficiently as opposed

to grain spawn. Liquid culture technology can increase the level of process control growth

rates and nutritional content (Friel & McLoughlin, 1999). Based on Leatham and Griffin

(1984) the process of spawn production in liquid culture production should rapidly and

reliably inoculate the given substrate. In order to produce a high quality liquid culture it

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should have a high density of viable inoculum particles. At the same time, the culture

should be homogeneous and consists of unpelleted mycelium (Itavaara, 1993).

There are many studies that have been done on liquid spawn production using the

submerged method. Kawai et al. (1996b) applied the liquid spawn method of L. edodes

on a sawdust fruiting substrate. Friel and McLoughlin (2000) has done submerged method

in the production of A. bisporus liquid spawn such as under static condition, continuous

incubation under shaking at 3000 rpm and incubation by exposure to alternating period

of agitation at 100 rpm to 300 rpm. Corfortin et al. (2008) produced Pleurotus sajor-caju

biomass in submerged culture, combining soy protein, yeast extract and ammonium

sulfate. Abdullah et al. (2013) produced high amounts of P. pulmonarius biomass in

brown sugar, rice bran, malt extract and yeast extract medium using an automated

bioreactor. At the same time, liquid spawn has been broadly used in the cultivation of

many species of mushrooms, including oyster mushroom (Alain, 1966; Silveira et al.,

2008), L. edodes (Kirchhoff & Lelley, 1991; Pellinen et al., 1987), Pleurotus

ostreatoroseus (Rosado et al., 2002), A. bisporus var. hortensis, Tricholoma nudum,

Morchellahortensis, Morchella esculenta and Cantharellus cibarius (Alain, 1966).

Despite these facts, there are some disadvantages by using liquid spawn in

mushroom cultivation. It is strenuous to store and transport liquid spawn and its residual

nutrients may cause contamination (Kumar et al., 2017). Additionally, liquid spawn of

several mushroom (A. bisporus and L. edodes) could germinate on some cultivation

substrate (Friel & McLoughlin, 1999; Leatham & Griffin, 1984). According to Friel and

McLoughlin (2000), the liquid spawn without nutrition, could not survive in pasteurized

compost and that the biomass levels were significantly lower than that of conventional

grain spawn with the entrapment of both mycelium and nutrients. In order to improve the

liquid spawn method, the aseptically encapsulated mycelium (EM) were implemented in

spawning mushroom production.

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Meanwhile, in the agriculture industry, immobilization technique has been

extensively used in the preparation of inocula to improve its ecological competence

(McLoughlin, 1994; Harith et al., 2014; Kumar et al., 2017). By using this technique, the

EM had a higher growth rate in pasteurised compost than both liquid spawn and the

conventional grain spawn by a shorter adaptation (lag) period. Also, EM was associated

to the high biomass loading capacity of these beads and afford a high viable inoculum

offering protection of the mycelium (Friel & McLoughin, 1999; Harith et al., 2014).

Certain works on EM of edible mushroom have been reported. A study conducted

by Rosado et al. (2002) on button mushrooms showed that, the EM were prepared by

entrapping liquid spawn with vermiculite, hygramer, and nourishment in sodium alginate;

its form was similar to that of grain spawn in the shape and yield of fruiting body. In

another study performed by Wang et al. (2011), the P. ostreatus mycelium was

encapsulated by using a mixture of cottonseed hull, corn core and wheat bran with a ratio

of 4.5:4.5:1. The outcome of using this EM showed similar result as using the grain

spawn. A study done by Friel and McLoughin (1999) show a potential of EM of A.

bisporus in pasteurised compost. Ortiz et al. (2017) reported that EM of A. bisporus, L.

edodes, P. ostreatus and Gymnopilus pampeanus showed no significant difference when

compared to conventional spawn to germinate the mycelium on sterilized substrate. In

addition, they found that the EM can be preserved frozen for six months and the mycelia

remain viable in most of the species assayed.

2.2.2.3 Submerged fermentation of mycelium to produce liquid spawn and encapsulation medium

Typically, nutrient source plays an important role affecting the yield of any

submerged products (Ma et al., 2016). Many works reported that carbon and nitrogen

source usually play a significant part because these nutrients are directly linked with cell

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proliferation and metabolite biosynthesis (Fang & Zhong, 2002a; López et al., 2003; Ma

et al., 2016; Park et al., 2001; Tang & Zhong, 2002).

Commonly, mushroom mycelium grows throughout a broad range of carbon

source (Yang et al., 2003). According to Chang and Miles (2004), the sort of carbon

sources for mycelial growth are starch, glucose, fructose, maltose, mannose, sucrose,

pectin, cellulose and lignin. Chang et al. (2006) reported that brown sugar and lactose can

stimulate mycelium formation in submerged culture. It is also stated that, although the

main component of brown sugar is sucrose, the present of trace elements in brown sugar

initiate on the production of mycelium and polysaccharide than sucrose. Hamachi et al.

(2003) state that brown sugar is usually distinguished as the most readily applicable

carbon source for most mushrooms cultures because it consists of approximately of 94–

98.5% (w/w) sucrose and various types of non-sucrose components ranging from 1.5–6%

(w/w). Furthermore, Hawker (1950) had mentioned that contribution of brown sugar

being low cost and easily available in supply compared to glucose, sucrose and lactose is

more suitable for the preparation of mushroom growth medium on a large scale.

Many authors stated that organic nitrogen sources frequently gave higher mycelial

biomassn growth in submerged culture (Yang et al., 2003; Shih et al., 2006; Asatiani et

al, 2008). The type of organic nitrogen that be used in submerged are yeast extract,

peptone (Pokhrel & Ohga, 2007), yeast powder (Jr-Hui & Shang, 2006) and casein (Fang

& Zhong, 2002a). Jr-Hui & Shang (2006) stated the effect of nitrogen source on the

mycelia growth of higher fungi be influenced by on the species and cultivation conditions.

Brewers’ spent grains (BSG) are accessible at low or no cost throughout the year

and are generated in bulky quantities by both large and small breweries (Aggelopoulos et

al., 2013). Spent grain is rich in cellulose and non cellusolusic polysaccharides (Aliyu &

Bala, 2011). Therefore, spent grain needed extracellular enzymes to break down to

simpler, soluble units so that it is easier to absorb nutrition for fungal hypae growth

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(Chang et al., 1993). Gregori (2008), stated that the additional of high concentration of

spent grain leads to a decreased enzymes activity. In addition, spent grain caused

insufficient oxygen supply for maximum growth during fermentation (Nakano et al.,

1997). Roberts (1976) stated that use of spent grain extract in the fermenter acts as an

effective antifoaming agent. According to Stein et al. (1973) spent grain extract may

supply 30% to 60% of the biochemical oxygen demand. Furthermore, spent grain extract

contribute in aerobic biological organism by breaking down organic material present in

the medium and enhance the mycelial growth in submerged medium.

Tween 80 is a non-ionic surfactant that is effective in discharging fungi enzymes

to the external environment (Rancaño et al., 2003) and exhibits low toxicity to the cellular

membrane (Giese et al., 2004). Its efficacy alters the structure and the morphology of

fungi and bacteria cell wall, prominent to the enhance of protein secretion (Giese et al.,

2004). Furthermore, effect of Tween 80 in submerged medium is it has the capability to

boost the growth of the mycelia (Zhang & Cheung, 2011; Zhang et al., 2012). According

to Li et al. (2011), by adding Tween 80 in submerged medium it can achieve small pellets

and to produce unicellar propagates, viscosity of broth, increase oxygen transfer and

biomass. In addition, Tween 80 has the potential to hinder breakdown of mycelial cells

due to the shearing forces during the shake-flask experiments by maintaining the intact

structure of the mycelial pellets during fermentation (Zhang & Cheung, 2011). According

to Domingues et al. (2000), the effect of Tween 80 on mycelial morphology could be

associated in part to its surface-active properties, lowering the mycelium-liquid interfacial

tension and consequently the potential or tendency of mycelia to form aggregates. By

decreasing the surface tension of the medium by surfactant lowers the thermodynamic

potential for the aggregation but favours the dispersion of mycelia. A work done by

Zhang & Cheung (2011), used Tween 80 as stimulatory agent in the submerged

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fermentation of mushroom mycelium due to it competence maintained the pH value of

the fermentation broth at an acidic lever.

Encapsulation medium technique was improved by integration of nutrient

carriers/ supplementation such as wheat bran, milled chitin, corn cobs, fish meal, soy

fibers, peanut hulls, gluten (Chien et al., 2001) and casein (John et al., 2010) into the

biopolymers (e.g. alginate) to afford a nutrition source essential for propagation of the

microorganisms (El-Komy, 2001; Woodward, 1988). Various supplementations which

help in the stability and survival rate can be affected thru the formation and storage of

micro-beads (John et al., 2011). John et al. (2011) reported that, the supplementation in

encapsulated medium is used to preserve the viability of microorganisms or to uphold the

properties of the microcapsule also have significant effect on the performing of

microorganisms in microbeads. Supplementation are the ingredients that help in the

maintenance and protection of the microbial cells in a formulation thru storage, transport,

and at the target zone (Xavier et al. 2004). Also based on the works by Young et al. (2006)

reported that the encapsulated medium must be stable from manufacture to application

site, it would boost the activity of the organism in the field, be inexpensive, and be

practical. At the same time, beside with carrier, supplementations act a vital role in an

extended survival thru different phases of formulation.

Gluten is a natural polymer and the major by-product from the manufacture of

wheat flour. Gluten is beneficial for practice as the matrix for supplementation in

encapsulated medium of cells or cellular elements because it is biodegradable, low-cost,

nontoxic and instantly accessible (Chien et al., 2001). A study done by Chien et al. (2001),

were used gluten for entrapping fungal mycelia.

A study done by Bok et al. (1993) are using biopolymeric gels such as potato

starch, rice, rye, barley, and soybean powders for entrapped biopesticide. These is done

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to hold its entomocidal activity through better protection against dehydration, sunlight,

heat, and the damaging effects of UV light.

2.2.2.4 Spawn quality

Ortiz et al. (2017) stated that the vital factor that contributes to yield of mushroom

production is spawn quality. According to Chang and Miles (2004), some aspects that

need to be considered in production of quality spawn are:

i) the genetic potentials of the fruiting culture for vegetative growth both in the

spawn substrate as well as spawn running in fruiting substrate and for quality

mushroom production;

ii) the type of the spawn material because this stimulus the speed and

thoroughness of mycelial growth in the spawn substrate as well as spawn

running in the fruiting substrate;

iii) spawn substrate cost and accessibility;

iv) the ability of the spawn to survive while in storage;

v) a good quality spawn will encourage the growth rate of the mycelia and by

that the window of opportunity for contaminants is significantly restricted and

yield are increased. (Stamets, 2000).

2.2.3 Spawn running / Incubation

The inoculated substrate bag is then incubated at temperature 23 to 27⁰C.

Generally, incubation temperature runs higher than the temperature for primordia

formation (fruiting phase) (Stamets, 2000). Further, the environmental condition for

incubation is no direct sunlight, unfiltered or no bright light because light can be

damaging or harmful to the mycelial. This phase is referred to as the mycelial growth

stage, which develops immediately after inoculation (spawning) until the bag of substrate

is completely permeated with mycelium. Spawn running (mycelial running) is the phase

during which mycelium develop out from the spawn and permeates the substrate. During

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the spawn running, the mycelium secretes enzymes to degrade the complex substance in

the substrate of the bag and the mycelium assimilates and conglomerates the needed

nutrients in sufficient amounts required for fruiting (Shu-Ting & Philip, 2004).

2.2.4 Fruiting phase

The formation of fruiting bodies of mushrooms is commonly in rhythmic cycles

called “flushes”. Management of mushroom house is vital in this stage as it is in this stage

that the mushroom development phase begins. In order to obtain primordia formation,

followed by the formation of fruiting bodies, optimum environment condition is

necessary. This stage of environment condition is different from those for spawn running.

During this fruiting phase, we need to maintain the optimum temperature, humidity and

ventilation. These factors will influence the number of flushes and total yield that will be

obtained. Requirement of humidity, temperature and ventilation for fruiting is commonly

different than for mycelial running. In the meantime, inappropriate aeration will

contribute to an increase in carbon dioxide (CO2) in the vicinity of the mushroom beds

where the mycelium is respiring. Therefore, this may hinder formation of primordia or

later mushroom developmental stages.

Based on Sánchez (2010), mushrooms are ready to be harvested almost 3 to 4

weeks after spawning, although it is influenced by the strain, amount of supplement used,

and temperature of fruiting house.

2.3 Pleurotus pulmonarius

Pleurotus pulmonarius (Fr.) Quél commonly known as grey oyster mushrooms

has increased its popularity due to its high nutrition level and its delicious taste. The genus

Pleurotus has been intensively studied in many different parts of the world due to many

reasons: they have high gastronomic values, they are able to colonize and degrade a large

variety of ligno-cellulosic residues, they require shorter growth time when compared to

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other edible mushrooms, they demand few environmental controls, their fruiting bodies

are not very often attacked by diseases and pests and they can be cultivated in a simple

and cheap way (Jwanny et al., 1995; Patrabansh & Madan,1997). In addition, it improves

the physical properties of the soil or as feed for animal by converting the substrate to be

used as a fertiliser (Fox, 1993; Maziero & Zadrazil, 1994; Bononi et al., 1995). This edible

mushroom is also used in environmental remediation (Pérez et al., 2008; Yan et al., 2009),

and biofuel technology (Okamura-Matsui et al., 2003).

From the standpoint of its medicinal values, the compounds extracted from P.

pulmonarius are capable to treat/cure various types of diseases, including atherosclerosis

(Abidin et al., 2018), hypertension (Ajith & Janardhanan, 2007) and cancer (Xu et al.,

2014) since it possessed significant antioxidant, anti-flammatory and antitumor activities

(Badole et al., 2006). Research done by Reshetnikov et al. (2001) has proven that P.

pulmonarius is rich in pharmacologically active polysaccharides such as xyloglucan and

xylanprotien.

From the nutritional standpoint, this mushroom contains high quantities of

proteins, carbohydrates, minerals (calcium, phosphorus, iron) and vitamins (thiamine,

riboflavin and niacin) and low fat (Randive, 2012). According to Sharma and Madan

(1993), the genus is characterized by its high protein content 30- 40% on dry weight basis

which is twice that of vegetables. The content of niacin in oyster mushroom surpasses

almost ten times than any other vegetables and the folic acid content in oyster mushroom

is beneficial for conditions such as anaemia (Randive, 2012). Besides that, it has benefits

for people who have hypertension, obesity and diabetes, due to its low sodium: potassium

ratio, starch, fat and calorific content. The content of alkaline ash and high fibre in the

oyster mushroom give an advantage for those suffering from hyperacidity and

constipation and cholesterol inhibitors (Randive, 2012).

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Pleurotus spp. are known to be easily and most widely cultivated in various

agricultural wastes in the world (Chang, 1999a; Akyüz & Yildiz, 2008; Shauket et al.,

2012). It has a promising future in the tropical and subtropical countries in the range of

moderate temperature from 20 to 30°C with 55-70% of humidity, because it is easily

cultivated and has a relatively inexpensive cultivation technique (Chang & Miles, 2004;

Randive, 2012).

Figure 2.4: P. pulmonarius a & b) a mature fruiting body was cultivated on sawdust substrate.

Various technique has been implemented in the cultivation of Pleurotus spp., such

as in different bagging systems like trays, cylindrical containers, wooden or polystyrene

racks, blocks and plastic bags (Quimio et al., 1990). Zadrazil and Kurtzman (1982) has

reported that by using plastic bag in cultivation has improve in yield of harvesting with

reduced contamination rate. Commonly, growers in Europe uses large black perforated

bags, however Asian growers’ mostly favour the use smaller sized bags, where the

technique of inoculation and harvesting are handled at one end of the bag. Meanwhile,

mushroom production in Japan use polypropylene bottle technology by filling the bottles

with sterilised substrates and then they are inoculated mechanically after substrate is

cooled (Royse, 1995).

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In addition to sawdust substrate, various types of lignocellulosic substrates can be

used to cultivate Pleurotus spp., such as paddy straw (Chang & Quimio, 1982; Rani et

al., 2008; Ahmed et al., 2009), leaves (Zadrazil, 1978; Shah et al., 2004), cotton stalks

(Balasubramanya, 1981; Balasubramanya & Khandeparkar, 1989; Hüttermann et al.,

2000), coffee pulp (Chang & Quimio, 1982; Guzman & Martinez, 1986), coffee residue

(husks) (Fan et al., 2003); rice hulls, water hyacinth leaves, coconut shell, corncobs and

leaves, (Hadar et al., 1992), sugarcane bagasse (Chang & Quimio, 1982; Hadar et al.,

1992), tree leaves wood wastes of timber work shops, cupola of nut trees, corn stalks,

waste tea leaves of tea factories, and waste paper (Sivrikaya & Peker, 1999), rice straw

(Hüttermann et al., 2000; Cayetano-Catarino & Bernabé-González, 2008; Daba et al.,

2008), peanut shells, cotton waste (Philippoussis et al., 2001), sawdust, oil palm fibre,

dry cassava peels (Onuoha et al., 2009), banana leaves (Cayetano-Catarino & Bernabé-

González, 2008), banana pseudostem, sorghum stalk (Rani et al., 2008), cotton seed hulls

(Daba et al., 2008), soybean straw (Daba et al., 2008), wheat straw (Shah et al., 2004;

Daba et al., 2008; Ahmed et al., 2009), handmade paper and cardboard industrial waste

(Kulshreshtha et al., 2013). Based on Mikiashvili et al. (2006) oyster mushroom has the

capability to generate lignolytic and hydrolytic enzymes and consequently can be easily

cultivated for fruiting by using a type of lignocellulosic waste and by enhancing the

carbohydrates availability and biomass accumulation.

2.4 Cultivation of P. pulmonarius in Malaysia

Mushroom production in Malaysia is concentrated on fresh mushrooms. Ninety

percent of the growers concentrate in P. pulmonarius cultivation because of its popularity,

high demand and many products that have been developed from this mushroom.

According to the Department of Agriculture of Malaysia (2015) the total production of P.

pulmonarius was recorded to be 90.89%. Meanwhile, in the hotel trade and caterers are

demanding for the Shiitake and button mushrooms in the market (Haimid et al., 2013).

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The total value of mushrooms production in Malaysia has increased from RM49.1 million

in 2007 to more than RM110 million in 2014. (Department of Agriculture of Malaysia,

2015). The remarkable improvement of production value was influenced by the

increasing number of growers, land area and productivity (Department of Agriculture

Malaysia, 2015). Mohd-Syauqi et al. (2014) stated that this increase being partly due to

the growth of population and higher interests towards health. The Ministry of Agriculture

(2011) predicted that the consumption of mushrooms will increased at a relative rate from

1.0 kg/ person in 2008 to 2.4 kg/ person in 2020.

According to the Department of Agriculture of Malaysia (2015), the total amount

of growers in Malaysia has reportedly increased every year from 339 growers in 2007 to

428 growers in 2014. About 80% of these growers consists of small growers, with the

production of fresh mushrooms of below 50 kg per hectare per day. Meanwhile, 17% of

the growers consists of medium scale growers (producing 50-500 kg of mushrooms per

day) and 3% is made up of big scale industries (producing more than 500 kg of

mushrooms per day) (Haimid et al., 2013).

A study done by Rosmiza et al. (2016) showed there were numerous problems

and challenges were identified that could obstruct effective mushroom industry growth in

Malaysia. In Malaysia we are facing a problem in getting the raw materials supply with

increasingly expensive price, especially sawdust and rice bran. In the cultivation stage of

mushroom, the poor quality of spawn contributes to low production yield of mushrooms

and may cause green fungus diseases.

Meanwhile, the lack of skills and knowledge among the mushroom growers in

mushroom cultivation may affect the yield of mushrooms. Sometimes mushroom growers

cannot overcome the problems regarding mushroom contamination due to low knowledge

in mushroom biology. Low skills in marketing of mushroom sales also contribute to this

problem (Rosmiza et al., 2016). At the same time mushroom growers are facing the issue

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in marketing competition from neighbouring countries such as Thailand. Thailand can

offer low price of mushroom due to low cost of raw materials that are available in their

country and can produce high yield of mushroom.

Mushroom growers are also facing problems regarding mushroom marketing due

to the short shelf life of fresh mushrooms (Rosmiza et al., 2016). At same time, there are

also factors such as the lack of awareness from local markets regarding the benefit of

eating mushroom and the limitation of knowledge (recipes) in preparing mushroom

dish/cooking. The lack of mushroom products in the market is also another contributing

factor in the mushroom marketing problem to growers.

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CHAPTER 3.0

MATERIALS AND METHODS

3.1 Preparation of P. pulmonarius mycelial culture

Pleurotus pulmonarius was cultured from cleaned mushroom sporophore by

wiped outer surface of the fruitbody with a clean tissue may harbor bacteria and mold

spores. Then, the pileus and stipe was split to expose the interior tissue is free from

contaminated organisms, assuming the sporophore is not water logged. The sporophore

was placed on the table with the clean surface faced sideways. Then immediately a small

fragment of tissue was firstly cut using a sterile scalpel and then pinched using a sterile

forcep and transferred into the centre of Petri plate containing malt extract agar (MEA)

as prepared in Appendix A, 1.0. The plate was sealed with parafilm and incubated in 25˚C

incubator for seven days. (see Figure 3.1).

Figure 3.1: Preparation of mycelial culture by tissue transfer; a) Splitting the sporophore and stipe to expose interior tissue, b) Cutting a small fragment of tissue using a sterile scalpel, c) Pinching a piece tissue fragment using a sterile forcep, d) Transfering the tissue fragment into the centre of MEA.

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The fruiting body has been authenticated by mycologist and by molecular method

and was given a code KLU-M1234 and deposited in Mushroom Research Centre,

University of Malaya herbarium. Pure mycelial culture was coded as KUM61119 and

deposited in Mycology Laboratory culture collection. The stock culture was preserved on

MEA slants stored at 4 °C.

3.2 Optimization of growth medium formulation for mycelial growth of P. pulmonarius by submerged fermentation

A series of experiments for fermentation condition and medium composition were

done to optimise the best low-cost liquid medium for the submerged fermentation of P.

pulmonarius mycelium using shake flasks. For the formulation of liquid medium, the

components selected were brown sugar as the carbon source based on Hawker (1950).

Brown sugar was also selected due to being low cost and easily accessible in supply

compared to glucose, sucrose and lactose is more suitable for the in preparation of

mushroom medium production in large scale. Various yeast types were tested as the

nitrogen source together with the supplementation of brewery spent grain extract,

minerals and Tween 80. In all the experiments, dry weight of P. pulmonarius mycelial

biomass was measured to evaluate the mycelial yield.

3.2.1 Optimization of brown sugar concentration and yeasts concentration

This experiment was done using a total volume of 50-mL liquid medium

consisting of either 0.5% or 2% of brown sugar as carbon source and three types of yeast

as nitrogen source i.e Baker’s yeast, brewer’s yeast and yeast extract. The percentage of

yeasts investigated was 0.1% and 1% (w/w) and pH for each formulation was measured

using a pH meter. The combinations of brown sugar and yeasts types at various

concentrations to formulate the liquid medium is depicted in Table 3.1.

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Table 3.1: The percentage of carbon and nitrogen sources concentration in liquid fermentation medium.

Brown sugar (w/v) (%)

Brewer's yeast (w/v)

(%)

Baker's yeast

(w/v) (%)

Yeast Extract

(w/v) (%)

Spent Grain (w/v) (%)

pH reading

C:N ratio

0.5 0.1 − − 0.1 5.32 117.83

0.1 − − 1 5.00 29.40

1 − − 0.1 5.59 34.56

1 − − 1 5.37 21.59

− 0.1 − 0.1 5.39 71.17

− 0.1 − 1 5.83 25.84

− 1 − 0.1 5.84 24.21

− 1 − 1 5.82 19.82

− − 0.1 0.1 6.58 47.98

− − 0.1 1 6.15 24.55

− − 1 0.1 6.91 13.05

− − 1 1 6.68 12.91

2 0.1 − − 0.1 5.27 433.75

0.1 − − 1 5.04 80.60

1 − − 0.1 5.15 99.97

1 − − 1 5.2 54.44

− 0.1 − 0.1 5.19 241.85

− 0.1 − 1 5.67 72.81

− 1 − 0.1 5.49 46.63

− 1 − 1 4.94 37.04

− − 0.1 0.1 6.14 164.90

− − 0.1 1 5.97 64.68

− − 1 0.1 6.85 27.51

− − 1 1 6.62 24.60

250-mL Erlenmeyer flask containing 50-mL medium was sterilised at 121 °C for

20 min and cooled to room temperature. Pleurotus pulmonarius culture was initially

grown on MEA medium in a Petri dish for 7 days, and ten 5 mm-diameter plugs cut from

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the periphery of the colony were transferred into the Erlenmeyer flasks. Inoculated flasks

were incubated on a shaking incubator at 25 ± 2°C rotating at 150 rpm for 6 days. All

medium formulation experiments were performed in triplicate flasks.

Mycelium pellets formed were filtered using filter paper (Whatman No.1) after 6

days of incubation. Mycelial dry weight was measured after repeated washing with

distilled water and left to dry at 60°C overnight in oven until constant weight was

achieved. All results were expressed as mean ± standard deviation from triplicates data.

Statistic significant differences were determined by One-way ANOVA Duncan test with p

values

32

Table 3.2: The percentage concentration of supplementation of spent grain extract, and minerals in liquid fermentation medium.

3.2.3 Effect of pH of optimised growth medium

The optimised growth medium (2% BS, 1% YE, 1% SGE, 0.05% M, 0.5% Tween

80) was prepared with pH of the medium adjusted to 4.50 (±0.05), 5.00 (±0.05), 5.50

(±0.05), 6.00 (±0.05), 6.50 (±0.05), 7.00 (±0.05), using 1M of sodium hydroxide (NaOH)

and 1 M of sulfuric acid solution (H2SO4). Procedure for preparing this liquid medium

and data collection is as described in 3.2.1. Three replicates were prepared for each

formulation. All results were expressed as mean ± standard deviation from triplicates data.

Statistic significant differences were determined by One-way ANOVA Duncan test with p

values

33

mycelium were cut from an actively growing plate on malt extract agar 7 days was

inoculated into the flask medium. Pleurotus pulmonarius broth was cultured and

incubated at 25°C ± 2°C on shaker at 250 rpm for 6 days.

Then 10% (v/v) of MB was inoculated into the 2-L stirred tank bioreactor (STR)

with an operational volume of 1.5 L containing of 2% brown sugar, 1% baker’s yeast, 1%

spent grain extract, 0.5% Tween 80, 0.05% MgSO4, 0.05% KH2HPO4 and 0.05%

K2HPO4(Figure 3.2). The cultivation conditions in bioreactor were as follows:

temperature (28ºC), agitation speed (250 rpm), initial pH (5.5), and oxygen partial

pressure (30-40%).

Reducing sugar concentrations in the MB was determined by the 3, 5-

dinitrosalicylic acid (DNS) methods (Miller, 1959) and expressed as glucose equivalents

(described in Appendix A, 2.0). Twenty mL samples of the MB being collected every 12

hrs for analyses up to 84 hrs. The biomass was determined after filtration of 20 mL of the

broth through filter paper (Whatman No.1). The solids were washed with of distilled

water. The filters with mycelium were dried at 60°C oven until constant weight was

achieved. Three batches of fermentation were done and data were represented as average.

All results were expressed as mean ± standard deviation from triplicates data. Statistic

significant differences were determined by One-way ANOVA Duncan test with p values

34

Figure 3.2: Two litre stirred tank bioreactor containing of 10% (v/v) of liquid inoculum in optimised medium consisting of 2% brown sugar, 1% baker’s yeast, 1% spent grain extract, 0.5% Tween 80, 0.05% MgSO4, 0.05% KH2HPO4 and 0.05% K2HPO4.

3.4 Non-supplemented encapsulation of P. pulmonarius mycelial broth (MB)

The objective of this study was to determine the optimum harvesting time of

bioreactor for good viability of P. pulmonarius mycelium. MB of P. pulmonarius was

prepared as inoculum in 500-mL Erlenmeyer flaks containing 100 mL of liquid optimised

medium (w/v) medium. The sterile medium was then inoculated with 20 mycelial plugs

from a six-days-old colony grown in MEA and incubated at 25 ± 2ºC on a shaker at 150

rpm for 6 days.

MB of P. pulmonarius (100 mLs) was then inoculated at 10% (v/v) into the

bioreactor containing sterile optimised medium. The cultivation conditions of bioreactor

were set as follows: temperature (28ºC), agitation speed (250 rpm), pH (6.0), and oxygen

partial pressure (30-40%). Pleurotus pulmonarius mycelium was cultivated for 48 hrs and

60 hrs to evaluate the optimum harvesting time.

MB of P. pulmonarius obtained from the bioreactor was then encapsulated using

encapsulation solution consisting of 500 mL of 2.5% (w/v) sodium-alginate salt (Sigma-

Aldrich), 2% (w/v) malt extract and 4% (w/v) glucose autoclaved at 121°C for 20 minutes.

All solutions, materials and apparatus were initially sterilized for 20 min at 121°C. After

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the encapsulated solution was cooled, it was mixed with 1 litres of a MB at the ratio of

2:1 (MB: encapsulated solution). The mixture was lightly agitated until homogenous and

then slowly dripped into a beaker containing 200 mL of 0.25 M calcium chloride (CaCl2)

solution using a peristaltic pump with continuous stirring as shown in Figure 3.3. The

received gel beads were allowed to hardened in this solution for 15 minutes and then was

washed twice with 200 mL sterile distilled water (Figure 3.3). The beads were stored at

4°C before use.

Figure 3.3: The setup for MB encapsulation.

Pleurotus pulmonarius encapsulated MB (PPEMB) was stored in vials for five

days and viability was observed on day three to determine percentage germination of

PPEMB. Five replicates sawdust substrates in glass petri dish (Appendix A, 3.0) was

prepared. Viability of PPEMB was tested by inoculating ten pieces of PPEMB on each

sterile sawdust substrate in glass petri dish. All petri dishes were double-sealed with

plastic paraffin film. The data were represented as percentage of germination of PPEMB

in average.

To determine the growth rate of PPEMB, one PPEMB was inoculated at the centre

of each sawdust substrate in glass petri dish (five replicates). Mycelial growth in each

petri dish growth rate were determined by measuring the average diameter every day for

MB

0.25 M of CaCl2

solution

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one week. The average reading was plotted against time (day) to obtain the growth rate

in mm/day.

3.5 Optimization of P. pulmonarius encapsulated mycelial broth (PPEMB)

3.5.1 Optimization of encapsulation solution by supplementation with mashed potato and soluble starch at various concentrations.

Mashed Potato (Appendix A, 4.0) and soluble starch grade AR (Friendemann

Schmidt) were selected as additional nutrient supplementation in encapsulation solution

to extend viability to allow for germination on sawdust substrate. The encapsulation

solution was supplemented with different concentrations of mashed potato or soluble

starch (3%, 5% and 8% w/v), to determine percentage germination upon storage of

PPEMB. Procedure for preparing this encapsulation solution is as described in section

3.4. Nutrient supplemented PPEMB (NS-PPEMB) was stored in different vials for 3, 7,

14, 21, 28 days and viability was determined upon storage. Five replicates sawdust

substrates in glass petri dish (Appendix A, 3.0) were prepared. Viability of NS-PPEMB

was tested by inoculating ten beads of NS-PPEMB on each sterile sawdust substrate in

glass petri dish. All petri dishes were double-sealed with plastic paraffin film. The data

was represented as average percentage of germination of NS-PPEMB.

3.5.2 Effect of supplementation of 5% mashed potato and 5% soluble starch in encapsulation solution on sporophore yield

Based on the result obtained above, supplementation with 5% mashed potato and

5% soluble starch in the encapsulation solution that had showed highest percentange

germination of P. pulmonarius mycelium. Thus, these NS-PPEMB (5% mashed potato

and 5% soluble starch) were evaluated as spawn for cultivation by mycelial growth and

sporophore yield expressed as biological efficiency on sawdust as fruiting substrate in

plastic bags. Three replicate bags were prepared (Appendix A, 5.0). The procedure for

preparing this encapsulation solution is described in 3.4. The NS-PPEMB were stored in

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sterile distilled water for two weeks in vials. After the sterile fruiting substrate bag were

cooled to room temperature, 5 pieces of NS-PPEMB were inoculated into fruiting

substrate bag by using sterile spatula and done under aseptic condition.

Then, the inoculated bags were incubated at an ambient temperature of 25˚C

(±2˚C) until full spawn run completed. Mycelia growth during spawn run was determined

by measuring mycelium extension at 4 sides of the bag at 2-day intervals for 30 days. The

average reading was plotted against time (day) to obtain growth rate in mm/day.

Once spawn running completed, all bags were transferred to the experimental

room temperature (30-32°C) mushroom house equipped with misting system. Sufficient

air flow was provided by the surrounding neeting of the mushroom house. Fully colonised

bags were scratched on the top of bag and subjected to an environmental relative humidity

of above 85%. This was done by spraying water in the form of fine mist using a sprinkler.

This was done to promote initiate sporophore formation and to provide space for

sporophore to emerge. Harvesting was done as sporophore matured and was done over a

period of three weeks. The yield of sporophore were recorded for two flushes was

expressed as biological efficiency (BE) and was calculated as below:

Percentage biological efficiency (BE) = Grams of fresh sporophore produced x 100 Grams of dry substrate used

All results were expressed as mean ± standard deviation from triplicates data.

Statistic significant differences were determined by One-way ANOVA Duncan test with

p values

38

and consistency of material for preparation of encapsulation solution and further

evaluated. The SS-PPEMB prepared as previous was stored in different vials for 5 days

and 10 days under the following conditions:

i. C1: Stored in vial without water at room temperature (25ºC)

ii. C2: Stored in vial without water at 4ºC

iii. C3: Stored in sterile distilled water at room temperature (25 ºC)

iv. C4: Stored in sterile distilled water at 4ºC.

This study was done in four replicates glass petri dish of sterile sawdust substrate

and on four replicates sterile fruiting substrate bags for each formulation. The preparation

of sterile sawdust substrate in glass petri dish was the same as in the Appendix A, 3.0.

After sterile substrate in glass petri dish were cooled to room temperature, 10 pieces of

SS-PPEMB were put on it. The percentage of 10 pieces of SS-PPEMB that germinated

on each petri dish and viability of storage life SS-PPEMB were observed. Four replicates

of sawdust substrate in petri dish were done and data were represented as percentage of

germination of SS-PPEMB mycelium in average